New non-paralytic botulinum molecules for the control of pain

In both animals and humans life events such as accidents or surgical interventions can lead to uncontrollable pain that can persist long after the original injury. This is chronic pain, the treatment of which is yet to be resolved - 19% of adult Europeans suffer from chronic pain of moderate to severe intensity; less than 60% receive adequate pain relief. New treatments are required but to develop these requires new therapeutic approaches and a better understanding of what goes wrong in the pain pathways resulting in uncontrollable pain.

Using the results of our recent research on Botulinum molecules, together with our existing knowledge of pain mechanisms, we will selectively silence pain signals both in pain fibers from the skin and other body tissues as well as in specific nerve cells in the spinal cord. Botulinum molecule is a protein and neurotoxin produced by the bacterium Clostridium botulinum. Botulinum molecule can cause flaccid neuromuscular paralysis, a serious and life-threatening illness in humans and animals. Popularly known by one of its trade names, Botox, it is used for various cosmetic and medical procedures. However we have found a way to separate the paralysing effects of the botulinum molecule from its silencing effects on other neurons. By using this new synthetic chemistry we will target and silence specific populations of neurons reversibly and alleviate chronic pain states.

The experiments outlined in this application will allow us to move rapidly towards the clinic and to apply these new Botulinum derivatives to different types of chronic pain. For example it is possible to imagine that a single injection of one of our Botulinum constructs into a painful arthritic joint or over the spinal cord of a patient with bone cancer pain would ameliorate pain without generating numbness or paralysis.

We have utilized the power of botulinum-mediated neuronal silencing and retargeted the botulinum molecule away from neuromuscular junction but retaining its actions on specific peripheral and central neuronal populations. We recently developed a new protein-linking approach that allows design of re-targeted versions of botulinum neurotoxins thereby opening a way towards meeting the need for acute reversible neuronal silencing. Specifically, the botulinum molecule was split into two halves that were expressed in E.coli as innocuous recombinant parts. Following purification, the two halves were linked together within 30 min. The re-assembled botulinum molecule exhibited the same efficacy on central neurons and brain slices as the native botulinum neurotoxin but had significantly reduced paralytic activity. Our results suggest that the new botulinum versions can provide a unique tool in probing CNS functions and blocking excessive neuronal activity in chronic pain conditions. We propose to fully evaluate the non-paralytic botulinum molecules in various pain models and develop new neuropeptide receptor-directed versions for dissecting neuronal circuits responsible for generation and maintenance of chronic pain. We will use synthetic chemistry to derive new molecules, in vitro tissue culture screens for selective uptake of conjugates coupled with immunohistochemical identification of different types of neurons and assays to measure release of neuropeptides. We will then translate this information to inflammatory and neuropathic pain models and record efficacy after peripheral or central application of botulinum constructs using behavioural analysis of mechanical and thermal sensitivity.

Despite considerable progress in our understanding of pain mechanisms, chronic pain has remained an area of considerable unmet medical need. A recent survey revealed that one in five people suffers from chronic pain in Europe and that the pain had a serious impact on their working and social lives. Currently only around 40% of chronic pain patients get reasonable treatment benefit implying that a large number of people will experience a lifetime of debilitating pain. There is therefore a pressing need for better pain killing medicines to manage chronic pain. There are a number of new approaches to controlling pain derived from experimental work on animals and humans. The approach used here centers on the synthesis of new non-paralyzing Botulinum constructs that target specific pain signalling neurons either within the central nervous system or periferally without having a paralyzing effect on muscles. We believe that these constructs will form the basis for a new range of therapeutics that will give rapid, reversible pain relief without killing neurons and also will help us to investigate the underlying causes of chronic pain states.